Hemin-Induced Mast Cell-Extracellular Traps Impart Resistance to Therapy in a Sickle Microenvironment

Mast cell activation in the skin contributes to inflammation, neurogenic inflammation and hyperalgesia in sickle mice (Vincent et al., Blood 2013). However, the underlying cause of mast cell activation and challenges in inhibiting mast cell activation remain unclear. Hemolysis in sickle cell disease leads to excess free heme, which contributes to endothelial activation and neutrophil extracellular trap (NET) formation. We therefore hypothesized that free heme activates mast cells and extracellular trap formation in a sickle microenvironment. We examined hemin-induced activation in mast cells from the skin of HbAA-BERK control and HbSS-BERK sickle mice. Mast cells in culture were incubated with vehicle, or I ng/ml TNF-α for 4 hours, or 40 μM hemin for 2 hours, or with hemin for 2 hours after 2 hours of priming with TNF-α to create an inflammatory sickle microenvironment. Cells were stained with cell impermeable DNA dye SYTOX orange and cell permeable dye SYTO13 to visualize the extracellular DNA fibers. Incubation with TNF-α or hemin did not show significant eruption of DNA from the cell body. However, sickle mast cells incubated with both TNF-α and hemin showed distinct DNA containing fibers exploding from the cell body with the appearance of spider web like formation similar to NETs. The length of majority of individual fibers was more than 50 μm stretching to more than 500 μM, indicative of TRAP formation. This response was blunted in mast cells from control mice. Thus, mast cells in a sickle microenvironment are primed and activated, and further priming with cytokines such as TNF-α leads to hemin-induced mast cell extracellular trap (MET) formation. Sickle mast cells express significantly higher TLR4 and FcεRI as compared to control mice (Vincent et al., Blood 2013). Therefore, we examined if silencing of FcεRI and/or inhibition of TLR4 attenuated TNF-α primed/hemin-induced MET formation. TAK242 at a dose of 1 μM but not at 0.5 μM inhibited TNF-α primed/hemin-induced MET formation, but silencing of FcεRI had no effect. Since cannabinoids, imatinib, palmitoylethanolamide (PEA) and cromolyn are known mast cell inhibitors, we examined their effect on TNF-α/hemin-induced METs. Imatinib (30 - 100 μM), a known mast cell inhibitor, showed no significant effect, while cromolyn (100 μM) led to a modest decrease in MET formation. Cannabinoid CP 55,940 and PEA at a relatively low dose of 30 μM completely blocked MET formation. Functionally, imatinib, PEA and CP 55,940 reduced the release of cytokines TNF-α and RANTES from mast cells incubated with TNF-α/hemin for 24 hours. Hemin in the presence of TNF-α stimulated sustained activation of NLRP3 inflammasome signaling. Thus, mast cells in a sickle microenvironment are activated due to a rich inflammatory cytokine milieu, via activation of the inflammasome signaling upon stimulation with hemin. The unique phenomenon of MET formation is orchestrated by a combined action of inflammatory milieu and hemin, imparting resistance to common mast cell inhibitors such as cromolyn and imatinib. This new phenomenon of MET formation identified by us may in part contribute to challenges of treating sickle cell disease and other mast cell associated pathological conditions.